In the Phase I project, we demonstrated that a novel CD4-based protein, CD4-IgG2, has excellent properties for further development as a prophylactic agent for HIV-1 infection, to protect individuals from infection in settings such as vertical and occupational exposure to the virus. The major advantage of CD4-IgG2 over HIV immune globulin (HIVIG) and monoclonal antibodies to the virus is that CD4-IgG2 neutralizes all stains of HIV-1, including primary isolates of the virus. During the Phase I period, we demonstrated that CD4-IgG2 neutralized in vitro a panel of 28 primary HIV-1 isolates from different genetic clades. Moreover, CD4-IgG2 neutralized HIV-1 in undiluted viremic plasma in an ex vivo assay. Finally, we demonstrated that CD4-IgG2 protects hu-PBL- SCID mice from infection by primary HIV-1 isolates. This is the first demonstration of protection against primary HIV-1 isolates in an animal model using any immunotherapeutic or antiretroviral agent. In the Phase II project, we will continue the development of CD4-IgG2. First, we will scale-up the production process and generate one gram of the protein. We will test combinations of CD4-IgG2 with broadly neutralizing mAbs to HIV-1 for synergistic inhibition of the virus. Combinations will be analyzed in both a novel HIV-1 envelope glycoprotein-mediated membrane fusion assay and in an in vitro neutralization assay using primary HIV-1 isolates. We will then compare the protective ability of CD4-IgG2 alone versus the optimal CD4-IgG2-mAb combination in hu-PBL-SCID mice challenged with several primary HIV-1 isolates, and macaques challenged with simian-human immunodeficiency virus (SHIV) containing gp120/gp41 of a primary HIV-1 isolate. The development of a safe and effective prophylactic agent against HIV-1 is an urgent public health priority. The overall goal of the Phase II project is to determine whether CD4-IgG2, or combinations of CD4-IgG2 and broadly neutralizing mAbs, can protect against infection by primary isolates of HIV-1 in the best available animal models. If successful, the Phase II project will form the basis of clinical trials during the Phase III period. PROPOSED COMMERCIAL APPLICATION: Development of a CD4-IgG2 as a prophylactic agent against HIV-1 infection.